Procedure

DNA Prep

Best done the day before you plan to do your transformations. The backbone digest should run overnight (finishing the morning of the transformations). You can run the second round of PCRs on the day of the transformation, but I like to do them overnight as well (so they're ready the morning of).

Insert

Follow directions on Mutazyme kit

Ideal mutation rate will depend on target and screening strategy

DpnI treat the PCR product

Mutazyme can require large amounts of template. Adjust DpnI time accordingly

Clean up PCR product

Commercial kits work just fine here

Using PCR product as template, set up 8x 100uL PCRs using Pfu as the polymerase.

For a 2.5kb template, reamplification with Taq introduced roughly 0.6 nucleotide mutations per template. Switching to Pfu made this step effectively faithful.

Combine all 800uL of PCR product (generally 40-50μg of DNA) into one tube.

Backbone

Cut 8μg of vector backbone overnight.

Generally 400uL digest. Try to cut just inside the PCR primer binding sites. To be safe, I often add a third enzyme to cleave the middle of the insert.

Coprecipitation

Run a gel to check that your PCR and digest worked

Assuming success, make three tubes of DNA:

375uL insert + 150uL vector

375uL insert + 150uL vector

450uL water + 75uL vector (Negative control)

Phenol-chloroform extract and ethanol precipitate tubes (can't use commercial columns since it's so much DNA)

DO NOT resuspend - you'll use the cell suspension to resuspend the DNA later. Just leave the DNA precipitated in the tube

Transformation

Set up an overnight culture of your yeast strain (5mL is fine)

In the morning, dilute back to 50mL YPD at OD 0.1

Let the culture regrow to OD 1.3-1.5 (about 7 hours for me)

Meanwhile, if you have frozen Tris-DTT, thaw it on ice about an hour before you need it.

Also, chill your buffer E and the tubes holding the precipitated DNA

Add 500uL Tris-DTT, return culture to incubator for 10-15 minutes

Place an aliquot of YPD in the incubator now (to warm it for the transformations)

After each transformation, resuspend cells with 1mL warm YPD. Transfer to a 15mL falcon tube. Then wash cuvette with another 1mL YPD and add that to the tube. All four positive transformations can go into the same falcon tube.